T-cell lymphocytes play a critical role in cell-mediated immunity by providing for an adaptive response to specific pathogens. T-cell activation depends on activation of at least two signaling pathways, one that is antigen specific and the other that is antigen nonspecific. A T-helper cell antigenic response requires activation of a first signaling pathway by the binding of the T-cell receptor to an antigen bound to MHC (major histocompatibility complex) on the surface of an antigen presenting cell (APC). The antigenic response also requires activation of a second signaling pathway, which produces a co-stimulatory signal, by the binding of CD28 protein on the surface of the T-cell to CD80 (B7-1) and CD86 (B7-2) on the surface of the APC. Because the antigen-specific signal is not sufficient to generate a full antigenic response, T-cell receptor/antigen-MHC binding without co-stimulation may lead to clonal inactivation or anergy. (See, e.g., Jenkins et al., 1993, Curr. Opin. Immunol. 5:361). The co-stimulatory pathway plays a role not only in T-cell activation and differentiation, but also in tissue migration and peripheral tolerance induction. (See Salomon et al., 2001, Ann. Rev. Immunol. 19:225.)
Activated T-cells also express on their cell surface cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), a homologue of CD28 that binds CD80 and CD86 with higher affinity. CTLA4 expression results in competitive binding to CD80 and CD86, blocking the CD80/86-CD28 interaction and terminating T-cell activation. Because these signaling pathways determine the magnitude of a T-cell response to antigen, as well as downstream responses to antigen, agents that modulate one or more costimulatory signals, e.g., by blocking one or more of the interactions between CD80/CD86 and CD28 and/or CTLA4, may be effective in treating disorders that result from disregulated cell-mediated immune responses. (See, e.g., Mueller, et al., 1989, Annu Rev. Immunol. 7:445).
Suitable agents for the disruption of the CD80/86-CD28 interaction include antibodies and fusion proteins. For example, in an experimental autoimmune encephalomyelitis (EAE) model, T-cells were blocked using anti-CD80 and anti-CD86 antibodies. (See Racke, et al., 1995, J. Clin. Invest. 96:2195). In another example, anti-CD80 and anti-CD86 antibodies were used to block type 2 in vivo immune responses (i.e., immune responses mediated by IgG antibodies and directed against cell-surface or matrix antigens) and the production of IL-4. (Greenwald, et al., 1997, J. Immunol. 158:4088) CD28-Ig fusion proteins have been demonstrated to interact with CD80 and/or CD86 on dendritic cells and to activate them, which results in an adjuvant activity of soluble CD28 that produces enhanced T-cell mediated immune responses in vivo. (Orabona et al., 2004, Nature Immunol. 5(11):1134). Alternatively, CTLA4-Ig fusion proteins have been used to disrupt CD80/86-CD28 interactions. (See, e.g., McIntyre, et al., 2005, Drugs of the Future, 30:873).
Recent studies of T-cell activation and the costimulatory pathway indicate that CD80 and CD86 can play different roles in immune responses, and especially in autoimmunity. Thus, blocking the binding of only one of CD80 or CD86 to CD28 has been shown to produce disparate effects in vivo, depending on the disease being studied and the timing of administration (See Salomon, 2001, Annu Rev. Immunol. 19:225; Kuchroo, et al., 1995, Cell 80:707; Racke, et al., 1995, J. Clin. Invest. 96:2195; Lenschow et al., 1995, J. Exp. Med. 181:1145; and Greenwald, et al., J. Immunol., 1997, 158:4088).
Antibodies that bind to CTLA4 and disrupt CTLA4 inhibition of CD28-mediated T-cell responses are currently in development for the treatment of various cancers. Ipilimumab, a human anti-CTLA4 IgG1 antibody, is in Phase III trials for melanoma and in Phase II trials for breast cancer and prostate cancer. Tremelimumab, a human anti-CTLA4 IgG2 antibody, is in Phase II trials for prostate cancer, bladder cancer, and colorectal cancer.
Fusion proteins that bind to CD80 and/or CD86 and disrupt the interaction with CTLA4 and CD28 are good candidates for the development of immunomodulatory agents. Fusion proteins allow for the engineering of proteins with, for example, improved solubility, stability, deliverability, and/or activity. One such immunomodulatory agent is the soluble fusion protein abatacept (Orencia®), developed for the treatment of rheumatoid arthritis (RA) and juvenile idiopathic arthritis, autoimmune disorders which attack the joints, causing painful swelling that can eventually result in bone erosion and joint deformity.
Abatacept is a CTLA4-Ig fusion protein consisting of the extracellular domain of human CTLA4 and a modified Fc portion of human immunoglobulin G1 (IgG1) that includes hinge, CH2, and CH3 domains. The CTLA4 domain provides binding activity and the Fc portion is believed to provide stability, solubility and deliverability. Abatacept inhibits the interaction of CD28 on T-cells with CD80 and/or CD86 proteins on APC cells, thereby resulting in inhibition of T-cell activation. (See, e.g., Schwartz, 1992, Cell 71:1065). Accordingly, administration of abatacept results in a decrease in T-cell proliferation, activation and signaling, and attenuates the autoimmune response in rheumatoid arthritis. A second-generation soluble recombinant CTLA4-Ig fusion protein known as belatacept differs from abatacept in two amino acids and has a greater binding affinity for CD80 and CD86. Belatacept is in ongoing Phase III trials for use in kidney and other organ transplantation. (See Linsley et al., 2009, Immunol. Reviews 229:307-321). A third fusion protein, MAXY-4, a protein derived from abatacept but having increased binding to CTLA4 targets, is currently in preclinical development by Perseid Therapeutics, LLC and Astellas Pharma, Inc. for treatment of autoimmune diseases and transplant rejection.
Therapy with abatacept can be cost prohibitive. Higher affinity variants of abatacept could reduce the required effective therapeutic dose and therefore reduce the cost of therapy. Moreover, because less of the agent is required to achieve a beneficial result, the risk of developing an immunogenic response to the agent itself may also be decreased. Other side-effects may also be reduced. There is a need to provide improved fusion proteins with differential binding to the CD80 and CD86 proteins, for example, by generating variants with higher relative affinity for one isoform relative to the other. Such improved variants may provide targeted immune modulation for specific conditions or pathologies. Adverse effects or exacerbation of immune responses observed in some cases of CTLA4-Ig and anti-CTLA4 antibody therapies may also be reduced.
Citation or identification of any reference in Section 2 or in any other section of this application shall not be construed as an admission that such reference is available as prior art to the present disclosure.